Biotechnology

Question 1

What is the innate immune defence of bacteria?

Answer 1

Restriction Enzymes

Question 2

Restriction Enzymes…

Answer 2

…cut at a specific recognition sequence which is usually a 6-base palindrome

• Hydrogen bonds form between complementary sticky ends

Question 3

DNA Ligase…

Answer 3

… the enzyme that form the sugar phosphate phosphodiester bonds

• Like the glue enzyme

Question 4

What is the adaptive immune defence in bacteria?

Answer 4

CRISPR-Cas-9

Question 5

How is CRISPR-Cas 9 different from a R.E?

Answer 5

It can change the location is cuts depending on the sequence of the gRNA it contains

Question 6

How does CRISPR-Cas 9 locate match to gRNA

Answer 6

The CRIPSR-Cas 9 complex locates a PAM sequence, opens up the DNA, and looks for a match to gRNA

Question 7

Uses of CRISPR-Cas 9 in gene editing

Answer 7

1) Knock in a gene
- Damages gene so it no longer
works

2) Knock out a gene

Question 8

How do you knock out a gene?

Answer 8

When DNA is broken or cut, enzymes will try to fix the damage. However, this process can result in mutations, which can be used to knock out a gene

• Non-homologous end joining

Question 9

How do you knock in a gene?

Answer 9

You combine Cas 9 and a piece of DNA with ends that match the cells DNA, but with a middle segment that is a foreign gene.
The cell will repair with this foreign gene in it.

Question 10

Why must RNA be converted to DNA in PCR?

Answer 10

Needs to be double stranded

Question 11

How is sgRNA designed?

Answer 11

• Designed by identifying the gene that acts as the template
• Designed to be complementary to target sequence
• sgRNA forms with tracerRNA and Cas9 to find desired DNA target sequence

Question 12

DNA ligase…

Answer 12

…joins breaks in the phosphodiester backbone of DNA

Question 13

Are complementary sections cut using RE’s similar to primers in PCR? Explain

Answer 13

Yes.
- Complementary sequences are able to anneal to DNA on either end of target genes
- Primers have complementary sequences to the DNA on either end of the target DNA in PCR

Question 14

Benefits of not using foreign species for CRISPR editing

Answer 14

• Reduces risk of disease
• Reduces unknown risks
• Increases public perception

Question 15

What might a gene that enhances the growth of a plant exposed to any light, code for?

Answer 15

• Accessory pigment
• Protein that enables more light to be absorbed

Question 16

Name variables controlled when completing gel electrophoresis

Answer 16

• Amount of DNA (volume)
• Same dye
• DNA run for same amount of time
• Same person loading
• Same agarose concentration

Question 17

Outline the process and the roles of four different ‘genetic tools’ involved in creating the DNA fragment containing the human insulin gene with the connected β-gal enzyme gene and the insertion of this fragment into the plasmid.

Answer 17

• DNA ligase
• Restriction Enzymes
• PCR
• Gel Electrophoresis
• Restriction enzymes are used to cut open the plasmid to produce sticky ends.
• The human insulin gene is inserted into the plasmid and fixed by using DNA ligase.
• Use gel electrophoresis to isolate the recombinant plasmids.
• Use PCR to amplify the recombinant plasmids.